Biomedical subjects

Y Reiter

Publications and source records attributed to Y Reiter.

29 records · Page 2Linked to original sources

PubMed1994

Cytotoxic and antitumor activity of a recombinant immunotoxin composed of disulfide-stabilized anti-Tac Fv fragment and truncated Pseudomonas exotoxin.

Disulfide-stabilized Fv (dsFv)-immunotoxins are recombinant immunotoxins in which the inherently unstable Fv moiety, composed of the VH-VL heterodimer, is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. Anti-Tac(dsFv)-PE38KDEL is composed of such a dsFv, directed to the alpha subunit of the IL2 receptor (IL2R), and containing a truncated form of Pseudomonas exotoxin (PE38KDEL). We have found this new type of immunotoxin to be indistinguishable in its in vitro activity and specificity from its single-chain immunotoxin counterpart, anti-Tac(Fv)-PE38KDEL. We have now examined the therapeutically relevant factors, including stability, pharmacokinetics, and antitumor activity of this new disulfide-stabilized Fv-immunotoxin. We found that anti-Tac(dsFv)-PE38KDEL was specifically cytotoxic to human activated T-lymphocytes in addition to IL2R bearing cell lines. Anti-Tac(dsFv)-PE38KDEL was considerably more stable at 37 degrees C in human serum and in buffered saline than the single-chain immunotoxin, anti-Tac(Fv)-PE38KDEL. The half-life in blood was similar for both immunotoxins (approx. 20 min). The therapeutic potential of the disulfide-stabilized immunotoxin was evaluated using an animal model of immunodeficient mice bearing subcutaneous tumor xenografts of human IL2R-bearing cells. Anti-Tac(dsFv)-PE38KDEL caused complete regression of tumors with no toxic effects in mice. Because dsFv-immunotoxins are more stable and can be produced with significantly improved yields compared to scFv-immunotoxins, dsFv-immunotoxin may be more useful for therapeutic applications.

ADP Ribose Transferases↗

PubMed1994

Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulfide-stabilized Fv fragment.

Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. We have recently described a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin (PE38KDEL). This disulfide-stabilized immunotoxin is indistinguishable in activity and specificity from its single-chain immunotoxin counterpart (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 90: 7538-7542, 1993). We have now constructed and evaluated the stability, pharmacokinetics, and antitumor effect of a very similar disulfide-stabilized immunotoxin B3(dsFv)-PE38. This immunotoxin is specifically cytotoxic to human cancer cell lines such as A431 that express the B3 antigen on their surface. In addition, the dsFv-immunotoxin is more stable at 37 degrees C in human serum than the corresponding single-chain immunotoxin B3(Fv)-PE38. The survival of the disulfide-stabilized immunotoxin in the circulation of mice was determined by a bioassay on cultured A431 cells after administering the immunotoxin i.v. The half-life in blood was 23 min. To determine the therapeutic effects of the disulfide-stabilized immunotoxin, it was given i.v. to immunodeficient mice bearing s.c. human epidermoid carcinomas. The dsFv-immunotoxin caused complete regression of tumors with no toxic effect on mice. The antitumor effect was similar to that reported for the single-chain Fv-immunotoxin. Our data show that dsFv-immunotoxins retain full in vitro as well as in vivo activity when compared to scFv-immunotoxins. Because dsFv-immunotoxins have full activity, are more stable, and can be produced with significantly improved yields compared to scFv-immunotoxins, the dsFv-immunotoxins may be more useful for therapeutic applications than scFv-immunotoxins.

Animals↗

PubMed1994

Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions.

Disulfide-stabilized Fv's (dsFv's) are recombinant Fv fragments of antibodies in which the unstable variable heavy (VH) and variable light (VL) heterodimers are stabilized by disulfide bonds engineered at specific sites that lie between structurally conserved framework positions of VH and VL. We have recently described one example of a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin [Brinkmann, U., Reiter, Y., Jung, S.-H., Lee, B., & Pastan, I. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7538-7542]. This disulfide-stabilized immunotoxin has the same cytotoxic activity and specificity as its single-chain immunotoxin counterpart. To determine whether the stabilization of Fv's by disulfides at these positions is generally applicable, we made and analyzed two other dsFv-containing immunotoxins. One is made from the e23 antibody, which binds to the carcinoma-associated antigen erbB2; the other is made from the anti-Tac antibody, which binds to the p55 subunit of the IL-2 receptor. Comparison of the specificity and activity of these immunotoxins with those of their scFv counterparts revealed that e23(dsFv)-PE38KDEL was considerably more active than e23(Fv)-PE38KDEL, whereas anti-Tac(dsFv)-PE38KDEL was only somewhat more active than its single-chain counterpart. These results suggest that dsFv's have at least the same binding properties as scFv's, and in some cases they may have better binding. Thus, it should be feasible to use the positions we have identified in the conserved framework region to disulfide-stabilize many different Fv's.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP Ribose Transferases↗

PubMed1994

Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv.

Using molecular modeling technology, we have recently identified two positions in conserved framework regions of antibody Fv fragments (Fvs) that are distant from CDRs, and potentially can be used to make recombinant Fv fragments in which the unstable VH and VL heterodimer is stabilized by an interchain disulfide bond inserted between structurally conserved framework positions. A disulfide bond has been introduced at one of these positions, VH44-VL105, and shown to stabilize various Fvs that retain full binding and specificity. Recombinant immunotoxins, e.g. B3(dsFv)-PE38KDEL in which this disulfide-stabilized Fv moiety is connected to a truncated form of Pseudomonas exotoxin (PE; PE38KDEL) which contains the translocation and ADP ribosylation domains, are indistinguishable in binding and specificity from its single-chain immunotoxin counterparts. We have now analyzed the alternative position, (VH111-VL48), predicted by the modeling methodology, for disulfide stabilization of mAb B3(Fv) by producing a recombinant immunotoxin with such disulfide-stabilized (ds) Fv. This immunotoxin was also very active and retained full specificity to B3 antigen-positive cells. However, it was 2- to 3-fold less active than the VH44-VL105 dsFv-molecule. We also tested various biochemical features of VH44-VL105 and VH111-VL48 dsFv immunotoxins and compared them with the corresponding single-chain immunotoxin. We found the dsFv immunotoxins were more stable in human serum and more resistant to thermal and chemical denaturation than the single chain (sc) Fv immunotoxin. Because dsFv immunotoxins and dsFvs have full activity and specificity and improved stability, they may be more useful than scFv immunotoxins as therapeutic and diagnostic agents.

ADP Ribose Transferases↗

PubMed1993

A recombinant immunotoxin containing a disulfide-stabilized Fv fragment.

B3(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv region of monoclonal antibody B3 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the unstable Fv heterodimer (composed of heavy- and light-chain variable regions) is held together and stabilized by a disulfide bond [termed disulfide-stabilized Fv (dsFV)]. A computer modeled structure of the B3(Fv), made by mutating and energy minimizing the amino acid sequence and structure of McPC603, enabled us to identify positions in conserved framework regions that "hypothetically" could be used for disulfide stabilization without changing the structure or affecting antigen binding. This prediction was evaluated experimentally by constructing a disulfide-linked two-chain dsFv-immunotoxin that was produced in Escherichia coli. The activity and specificity of this immunotoxin was indistinguishable from its single-chain Fv (scFv) counterpart, indicating that, as in B3(scFv), the structure of the binding region is retained in B3(dsFv). Because we introduced the stabilizing disulfide bond in between two framework residues in a position that is conserved in most Fv molecules, this method of linkage between the heavy- and light-chain variable regions should be generally applicable to construct immunotoxins and dsFv molecules using other antibodies. Furthermore, the finding that B3(dsFv) was much more stable at 37 degrees C in human plasma than B3(scFv) indicates that dsFvs are possibly more versatile for therapeutic application than scFvs.

ADP Ribose Transferases↗

PubMed1992

Sublytic complement attack protects tumor cells from lytic doses of antibody and complement.

Sublytic doses of the membrane attack complex (MAC) of complement are known to exert multiple stimulatory effects on metabolically active cells. Results presented herewith demonstrate that pretreatment of the human leukemic cells K562 and HL-60 with sublytic doses of antibody and normal human serum protects them from lytic complement concentrations, a phenomenon proposed to be called "complement-induced protection". C7- and C8-deficient human sera are ineffective in inducing resistance unless they are reconstituted with purified human C7 and C8, respectively. The complement-induced protection is inhibitable by actinomycin D and cycloheximide indicating that the increased complement resistance depends on RNA and protein synthesis triggered by the sublytic complement doses. Free extracellular Ca2+ is also required to achieve maximal protection, indicating a role for Ca2+ ions in the cell stimulatory events which culminate in increased complement resistance. Quantitative analysis of bound complement components indicated that similar amounts of C3 and C9 molecules are deposited on "protected" and control cells during complement activation. The "protected" K562 and HL-60 cells regain sensitivity to lytic MAC doses after about 8 or 3 h, respectively, of culture in growth medium, in the absence or presence of actinomycin D and cycloheximide. The "induced protection" is not species restricted and protection from human complement can be induced in K562 cells by treatment with sublytic doses of antibody and rabbit or guinea pig sera.

Animals↗

PubMed1992

Complement membrane attack complexes induce in human leukemic cells rapid expression of large proteins (L-CIP).

The effect of sublytic doses of the complement membrane attack complexes (MAC) on protein synthesis in human leukemic cells was examined. As shown herein, rapid protein synthesis is evident in K562 erythroleukemic cells upon exposure to sublytic complement doses. Analysis of cell extracts by SDS-PAGE revealed high molecular weight proteins which appeared in the cells already after 15 min treatment with complement at 37 degrees C, reaching a maximal level after 40-50 min. These large complement-induced proteins (L-CIP) were clearly observed in gels stained by Coomassie blue and in autoradiograms following [35S]-Met or [3H]-Leu incorporation. Rabbit antibodies prepared against L-CIP were reactive in immunoassays with extracts of MAC-treated cells but not of non treated cells. They also bound to the surface of intact K562 cells (as determined by immunofluorescence), but only after treatment of the cells with complement. Both heterologous (rabbit and guinea pig) and homologous (human) sera induced L-CIP synthesis. The induction of L-CIP was indeed mediated by the complement MAC since L-CIP could not be detected in K562 cells exposed to heat-inactivated human serum or C6-deficient rabbit serum. Similarly, C7- or C8-deficient human sera could not induce L-CIP production unless they were reconstituted with purified human C7 or C8, respectively. The synthesis of L-CIP was largely inhibited by the protein synthesis inhibitors cycloheximide and puromycin and partially inhibited by the RNA synthesis inhibitor actinomycin D. L-CIP was similarly induced in two other human leukemic cell lines, U937 and HL-60, but not in K562/S, a subline of K562 which is highly sensitive to complement damage. These results are discussed with respect to the resistance of leukemic cells, and nucleated cells in general, to complement-mediated immune damage.

Cell Death↗

PubMed1991

Resistance to NK cell-mediated cytotoxicity (in K-562 cells) does not correlate with class I MHC antigen levels.

Natural Killer (NK) cells probably function as an early line of defense against virus-infected cells and tumor cells. In all cases, the killing by NK cell-mediated cytotoxicity (NK-CMC) is not MHC-restricted and the factors which determine the sensitivity to NK-CMC have not yet been identified. A positive correlation between resistance to NK-CMC and the level of class I MHC antigen (MHC I) expression on target cells has been reported in many studies, and in some cases a functional linkage between the two has been claimed. Several other studies have shown that there is no such correlation. By employing several experimental systems, we demonstrate here a lack of correlation between the level of MHC I and the sensitivity of K-562 cells to NK-CMC. Transfer of MHC I to MHC I-negative cells via vesicles had no effect on their resistance to NK-CMC. In addition, a decrease in resistance to NK-CMC and increase of MHC I levels was observed following target-cell membrane modulation by both application of cholesterol and hydrostatic pressure. Finally, no correlation between sensitivity to NK-CMC and MHC I expression was found in three sublines of K-562 cells. Since NK-CMC is a multistage process, it is concluded that components other than class I MHC antigens have a more prominent role in modulating the sensitivity of target cells to NK-CMC.

Cell Death↗

PubMed1990

Potentiation of NK cytotoxicity by antibody-C3b/iC3b heteroconjugates.

The interaction of two Burkitt lymphoma lines, Raji and Rael, with human C and NK cells was analyzed. Raji cells activate the alternative C pathway (ACP) and then bind C3 fragments. Consequently, the cells become more sensitive to lysis by CR3-bearing NK cells but not to C lysis. In contrast, Rael cells are poor ACP activators, do not bind C3 fragments, and are therefore resistant to C-dependent NK lysis. As suggested earlier, the difference between Raji and Rael could be attributed to the presence or absence of CR2, respectively, on their surface. To potentiate C- and NK-dependent lysis of target cells, we generated heteroconjugates composed of a murine antitransferrin receptor mAb and of human C C3b or iC3b. Antibody-C3b conjugates induced C3 deposition on Rael cells and elevated C3 deposition on Raji cells in human serum. Both Raji and Rael cells coated with antibody-C3b conjugates were efficiently lyzed by the cytolytic ACP in human serum. This conjugate had a small enhancing effect on target cell lysis by NK cells which could be markedly increased by combined treatment of the target cell with antibody-C3b conjugate and C5-depleted human serum. On the other hand, antibody-iC3b conjugates efficiently potentiated lysis of target cells by NK cells in the absence of serum. The iC3b-directed cytotoxicity was mediated by CR3-bearing NK effector cells. Anti-C3 but not anti-mouse Ig antibodies abrogated the activity of the antibody-iC3b conjugate. These results further demonstrate that NK cytotoxicity may be potentiated by opsonizing the target cells with C3 fragments and suggest that antibody-C3b/iC3b conjugates could be potent tools for targeting and potentiation of the lytic action of both C and NK cells against tumor cells.

Antigen-Antibody Complex↗

PubMed1989

Targeting of complement to tumor cells by heteroconjugates composed of antibodies and of the complement component C3b.

Tumor cells have adapted several strategies which permit them to grow in an immunologically hostile environment. The C system can potentially destroy these cells; however, its action needs to be specifically potentiated on the surface of the tumor cells. To this end, a heteroconjugate composed of a mouse mAb and of the human C3b C component has been generated by using the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate. The two mAb which were used in this study are V1-10 and TIB219 which bind to the human and mouse transferrin receptors, respectively. The mAb-C3b conjugates were purified by gel filtration and were each composed of one mAb and one C3b. They bound to the human K562 and HL60 or mouse ALB1 cell lines and amplified the killing of these cells by C from 10 to 15% to 70 to 100%. Fresh normal human or mouse sera were used as a source of C. The mAb-C3b conjugates activated primarily the alternative pathway of C since only C3 and factor B but not C4 were cleaved in the sera. After disulfide-linking to the mAb, the C3b became highly resistant to inactivation by factors H and I, probably due to its reduced factor H binding capacity. On the other hand, the conjugated C3b bound factor B better than free C3b and produced more C3 convertases which expressed increased stability. These results suggest that mAb-C3b conjugates may serve as an effective tool for the specific activation of the cytolytic C system on selected cells. As such, they may be used in vitro or in vivo to target the autologous C to tumor cells or to lymphocytes and may promote tumor immunotherapy.

Animals↗

PubMed1989

Killing of human tumor cells by antibody C3b conjugates and human complement.

To potentiate the lytic action of complement on tumor cells, we have constructed heteroconjugates composed of monoclonal antibody and of the human C3b component of complement. The conjugates were formed efficiently using the heterobifunctional cross-linking reagent SPDP. The monoclonal antibody-C3b conjugate promoted the killing of K562 tumor cells by normal human serum. Treatment of the tumor cells with the monoclonal antibody and normal human serum resulted in 10-15% lysis. However, following pretreatment of the cells with antibody-C3b conjugates, their lysis by normal human serum increased to 70%. The conjugate activated selectively the alternative pathway of complement and the C3b component in the conjugate was highly resistant to cleavage and inactivation by the complement regulatory proteins Factors H and I. These results suggest that the coupling of C3b molecules to monoclonal antibodies anti-unique tumor antigens produces a potent complement-activating reagent which may act specifically on tumor cells and promote cancer therapy.

Antibodies, Monoclonal↗